Abstract
The interleukin-13 receptor alpha2 (IL13Rα2) is a cell surface receptor that is over-expressed by a subset of high-grade gliomas, but not expressed at significant levels by normal brain tissue. For both malignant and non-malignant cells, IL13Rα2 surface expression is reported to be induced by various cytokines such as IL-4 or IL-13 and tumor necrosis factor (TNF). Our group has developed a therapeutic platform to target IL13Rα2-positive brain tumors by engineering human cytotoxic T lymphocytes (CTLs) to express the IL13-zetakine chimeric antigen receptor. We therefore sought to investigate the potential of cytokine stimulation to induce IL13Rα2 cell surface expression, and thereby increase susceptibility to IL13Rα2-specific T cell killing. In the course of these experiments, we unexpectedly found that the commercially available putative IL13Rα2-specific monoclonal antibody B-D13 recognizes cytokine-induced VCAM-1 on glioblastoma. We provide evidence that the induced receptor is not IL13Rα2, because its expression does not consistently correlate with IL13Rα2 mRNA levels, it does not bind IL-13, and it is not recognized by IL13-zetakine CTL. Instead we demonstrate by immunoprecipitation experiments and mass spectrometry that the antigen recognized by the B-D13 antibody following cytokine stimulation is VCAM-1, and that VCAM-1, but not IL13Rα2, is induced on glioma cells by TNF alone or in combination with IL-13 or IL-4. Further evaluation of several commercial B-D13 antibodies revealed that B-D13 is bi-specific, recognizing both IL13Rα2 and VCAM-1. This binding is non-overlapping based on soluble receptor competition experiments, and mass spectrometry identifies two distinct heavy and light chain species, providing evidence that the B-D13 reagent is di-clonal. PE-conjugation of the B-D13 antibody appears to disrupt IL13Rα2 recognition, while maintaining VCAM-1 specificity. While this work calls into question previous studies that have used the B-D13 antibody to assess IL13Rα2 expression, it also suggests that TNF may have significant effects on glioma biology by up-regulating VCAM-1.
Highlights
Malignant gliomas are highly aggressive and uniformly lethal human brain cancers for which tumor recurrence following conventional therapies remains a major challenge for successful treatment [1,2]
We investigated the possibility of up-regulating cell surface interleukin-13 receptor alpha2 (IL13Ra2) levels on glioma utilizing cytokine stimulation regimens previously reported to induce IL13Ra2 on other cell types [13,14,15], including the monocytic cell line THP-1 [13]
Using the commercially available PE-conjugated B-D13 mouse monoclonal antibody (B-D13-PE; Cell Sciences) reported to target IL13Ra2, we find that cell surface antigen expression on negative or low expressing glioma cell lines (T98, PBT003-4, PBT008 and PBT017-4) is up-regulated following incubation with either tumor necrosis factor (TNF) and IL-4, or TNF and IL-13 (Figure 1A)
Summary
Malignant gliomas are highly aggressive and uniformly lethal human brain cancers for which tumor recurrence following conventional therapies remains a major challenge for successful treatment [1,2]. Immunotherapy is emerging as a promising therapeutic approach due to its potential to seek-out and attack malignant cells, the infiltrated cells often responsible for disease recurrence, while sparing cells of the normal brain parenchyma For this reason, significant efforts are dedicated towards identifying targets amenable for immunotherapy of brain tumors. IL13(E13Y)-zetakine+ CTL retain MHC-independent IL13Ra2-specific anti-glioma cytolytic activity, maintain CAR-regulated Tc1 cytokine secretion and proliferation, and mediate regression of established human glioblastoma xenografts in vivo [12]. These pre-clinical studies have culminated in a FDA-authorized feasibility/safety clinical trial of intracranial adoptive therapy with autologous IL13-zetakine+ CD8+ CTL clones targeting recurrent/progressive malignant glioma
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
